Characterization of FimH adhesins expressed by Salmonella enterica serovar Gallinarum biovars Gallinarum and Pullorum: reconstitution of mannose-binding properties by single amino acid substitution

Abstract

Recombinant FimH adhesins of type 1 fimbriae from Salmonella enterica serovar Gallinarum biovars Gallinarum and Pullorum, in contrast to those of Salmonella enterica serovar Typhimurium, did not bind to high-mannose oligosaccharides or to human colon carcinoma HT-29 cells. However, mutated FimH proteins from biovar Gallinarum and biovar Pullorum, in which the isoleucine at position 78 was replaced by the threonine found in S. enterica serovar Typhimurium, bound well to glycoproteins carrying high-mannose oligosaccharides and colon carcinoma cells. The loss of sugar-binding properties by biovar Gallinarum and biovar Pullorum FimH adhesins, which are a part of the type 1 fimbriae, is most probably the result of a single T78I mutation, as was proven by site-directed mutagenesis of FimH proteins.

FIG. 1.

Immunodetection of FimA and FimH proteins of biovar Gallinarum (lanes 1, 3, and 5) and biovar Pullorum (lanes 2, 4, and 6). Type 1 fimbriae (10 μg), purified according to the method of Müller et al. (11), were dissolved in Laemmli sample buffer supplemented with glycine at pH 2.2 and subjected to SDS-PAGE under reducing conditions in 12% gel and electrophoretically transferred to nitrocellulose. The positions of the molecular mass standards (in kDa) are shown on the left. (A) SDS-PAGE gel stained with Coomassie brilliant blue; (B and C) Western blots stained with polyclonal chicken anti-FimA (B) and polyclonal rabbit anti-FimH (C) antibodies.

FIG. 2.

Coomassie brilliant blue-stained SDS-PAGE (A) and immunostaining (B) of recombinant FimH adhesins from biovar Gallinarum (lanes 1), biovar Pullorum (lanes 2), and Salmonella enterica serovar Typhimurium (lanes 3). FimH proteins (10 μg), purified on Ni-NTA resin, were subjected to SDS-PAGE under reducing conditions in 10% gel. The migration positions of protein standards (in kDa) are indicated on the left.

FIG. 3.

Western blot analysis of FimH binding to glycoproteins carrying high-mannose-type oligosaccharide chains. Binding of the recombinant FimH adhesins from Salmonella enterica serovar Typhimurium (A), biovar Gallinarum (B), and biovar Pullorum (C) to mannan-BSA (lanes 1), HRP (lanes 2), and RNase B (lanes 3). Individual glycoproteins (10 μg) were subjected to SDS-PAGE under reducing conditions in 10% gel and electrophoretically transferred to nitrocellulose. The positions of the molecular mass standards (in kDa) are shown on the left.

FIG. 4.

Western blot analysis of mutant FimH adhesins binding to glycoproteins carrying high-mannose-type oligosaccharide chains. Binding of the mutant FimH proteins from biovars Gallinarum and Pullorum in the absence (A and C) and presence (B and D) of 0.2 M d-mannose to mannan-BSA (lanes 1), HRP (lanes 2), and RNase B (lanes 3). The mutants, constructed by site-directed mutagenesis, are identical to wild-type adhesins except at the position corresponding to the amino acid at position 78, where isoleucine was replaced by threonine (I78T). Individual glycoproteins (10 μg) were subjected to SDS-PAGE under reducing conditions in 10% gel and electrophoretically transferred to nitrocellulose. The positions of the molecular mass standards (in kDa) are shown on the left.

FIG. 5.

Binding of serially diluted FimH adhesins (starting from 100 μg/ml) to a monolayer of human colon cancer HT-29 cells growing in 96-well plates. ▴, FimH from S. enterica serovar Typhimurium; ▪, native FimH from biovar Gallinarum; □, mutated FimH from biovar Gallinarum; •, native FimH from biovar Pullorum; ○, mutated FimH from biovar Pullorum.